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Reprinted from 
The Journal of Infectious Diseases; Vol. 37, No. 4, October, 1925, pp. 344-352 


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TOXIN PRODUCTION BY CLOSTRIDIUM 
BOTULINUM IN CANNED FOODS 


BY 


LUTHER THOMPSON 


B.A. Clark University, 1916 
M.A. University of Illinois, 1923 


UNIVERSITY of ILLINGIS 


A DIGEST OF A THESIS © 


SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS 
FOR] THE DEGREE OF (DOCTOR OF PHILOSOPHY IN 
BACTERIOLOGY IN THE GRADUATE SCHOOL 
_ OF THE UNIVERSITY OF ILLINOIS, 1925 


Reprinted from 
The Journal of Infectious Diseases; Vol. 37, No. 4, October, 1925, pp. 344-352 


Toxin Production by Clostridium Botulinum 
in Canned Foods 


Ib AO USdeoae IAS MORU Ea OMN 
WITH 
Pome Wee VIN) INGE 


Reprinted from 
THE JourNAL oF InFEcTious Diseases, Vol. 37, No. 4, Oct., 1925, pp. 344-352 


oO SNe ePROwUe TON Ey SCeOSTRIDIUM “BOTULINUM 
IN CANNED FOODS 


PweeEer ew oMP SONG WET H Ob. We LAN NER 


From the Department of Bacteriology, University of Illinois, Urbana 


This investigation was begun in the summer of 1923, and was pri- 
marily intended to test the ability of Clostridium botulinum to grow and 
produce toxin in canned foods of widely varying composition. It was 
hoped in this way to indicate those foods that are potentially dangerous, 
although some of them might never have been reported as the cause of 
food poisoning. In the fall of the same year the paper by Schoenholz, 
Esty and Meyer ? appeared, which treated in detail the problem and gave 
results corresponding essentially to those obtained in the present investi- 
gation. For this and other reasons the work was extended to another 
phase of the general problem, namely, the explanation of the irregularity 
of toxin production in certain foods. It often has happened that one 
or more cans of food from a lot have been poisonous, while the greater 
number of them have been harmless. ‘This is particularly true of vege- 
tables such as spinach, asparagus, beets, and canned greens. 


Although a number of experiments have been performed on inoculation of 
foods with Clostridium botulinum, few have been comprehensive in scope. For 
the most part from 1 to 3 or 4 kinds of food were used. Dickson? in showing 
that a meat medium is not necessary for growth and toxin production, inocu- 
lated cans of beans and peas as well as pork and beef, and found that all 
produced strong toxins. Dickson, Burke and Ward®* used cans of apricots, 
peaches, and pears inoculated with washed spores, and after incubation for 
3 months a toxin was present in such quantity that 1 cc. of the material 
injected into a guinea-pig would cause death in from 1 to 2 days. In the 
light of recent experiments these results are less convincing, since the inoculum 
was not detoxified by heat. Armstrong, Story, and Scott* used ground ripe 
olives as a medium, and stated that a strong toxin was produced in 9 days. 
Koser, Edmondson, and Giltner® inoculated cans of spinach with approxi- 
mately 800,000 washed, heated spores. They reported that toxin production in 
spinach was irregular. Of 174 cans of spinach selected from various com- 
mercial lots, 6 contained toxin. Bengtson® used string beans, peas, spinach, 
olives, corn, and beets as mediums. On all of these except beets and olives a 
strong toxin was produced. Here also heated spores were not used in every 
case. In studying the relation of toxin production to temperature, Edmondson, 


Received for publication, May 6, 1925. 


1 Jour. Infect. Dis., 1923, 33, p. 289. 

2 Jour. Am. Med. Assn., 1915, 65, p. 492. 
3 Arch. Int. Med., 1919, 24, p. 581. 

4 Pub. Health Rep., 1919, SAW Dee couse 

5 Jour. Am. Med. Assn., 1921, 77, p. 1250. 
6 Pub. Health Rep., 1921, 36, DP. 1665. 


4. L. THomMrpson AND F. W. TANNER 


Giltner, and Thom‘ inoculated peas in open flasks and bottles of milk. Their 
results show that a temperature below 20 C., and lack of anaerobic conditions 
may retard or prevent toxin production. The first comprehensive attempt to 
inoculate canned food was made by Schoenholz, Esty, and Meyer.” These 
investigators, using heated spore suspension, inoculated a large series of cans 
of foods comprising the following varieties: Group 1l—corn, peas, sweet 
potato, salmon; Group 2—sauerkraut, tomato, apricots, cherries, peaches, pears, 
plums, raspberries, strawberries; Group 3—asparagus, beets, olives, peppers, 
spinach, string beans, evaporated milk. Group 1 showed marked physical 
and chemical changes, and were regularly toxic; group 2 rarely showed 
spoilage or toxin, while group 3 was irregular, both as to signs of spoilage 
and toxin production, certain foods being very toxic, at times without physical 
signs of spoilage. For the most part, type A cultures were used for inoculation, 
each can receiving from 50 million to 17 billion spores, and the incubation of 
inoculated foods was continued up to 12 months. An attempt was made-to 
correlate the growth and toxin production with the hydrogen ion concentration 
of the foods at the end of the incubation period. Considerable variation in 
acidity was noted among cans of the same kind of food, and tables are given 
showing the relation between acidity and the ability of Clostridium botulinum 
to grow and produce a toxin. Edmondson, Thom, and Giltner® inoculated 
normal cans of spinach with toxin-free spores, and noted irregularity in growth 
at the same temperature. They state that some cans may be toxic while show- 
ing little physical evidence of spoilage. These authors’ also inoculated peas, 
milk, cream cheese, meat balls and mashed potato, which were placed in con- 
tainers to simulate the conditions in which they would naturally be kept in the 
household. It is interesting that a strong toxin may be produced under condi- 
tions which are not usually considered anaerobic, as for example in the middle 
of a meat ball, or in the bottom of a dish of mashed potato where no particular 
precautions are taken to exclude air. Bachmann” used as mediums chopped 
green beans and cabbage packed in test tubes. The beans developed a toxin, 
while the cabbage did not. Lynn and Sells,” in studying the chemical changes 
produced by Clostridium botulinum in food, used string beans as a medium. 


EXPERIMENTS 


The cultures used in our experiments comprised 2 type A strains known 
in this work as Al and A2, which were received from the Department of Health 
Laboratories, New York City, and came labeled as “Delbene” and “Bronfen- 
‘brenner,” respectively; also 2 type B strains, 175 B (Nevin) and 7870 (Graham). 
All strains have been typed from time to time and have always proved true to 
name. Before use they were tested for toxin production in canned corn; in 
all cases the cans developed into “hard swells” in 48 hours, and a potent toxin 
was formed such that when 1 cc. of the liquor was fed to guinea-pigs, the 
death time varied from 5 to 18 hours. 

These strains were grown in flasks of sugar-free casein digest broth, or 
coagulated egg broth, covered with paraffin, and. incubated at 37 C. until 
growth apparently had ceased, and the organisms were well sedimented. This 
varied with the strains from 30 to 60 days. The supernatant fluid was then 
decanted, and the sediment containing the spores was well washed in salt 


7 Abstr. Bacteriol., 1922, 6, p. 23. 

8 Am. Food Jour., 1923, 18, p. 143. 

® Ibid., p. 143. 

10 Jour. Infect. Dis., 1924, 34, \p. 129. 
11 Abstr. Bacteriol., 1924, 8, p. 31. 


Toxin PropucTION By CLOSTRIDIUM BoTULINUM 5 
2 

solution, suspended in 25 cc. of salt solution and heated in an oil bath at 80 C. 
for 15 minutes. The use of 150 cc. flasks for this purpose insured quick heat 
penetration due to the shallow layer of liquid. The spore suspensions were 
then shaken by machine with glass beads for 15 minutes to break up clumps, 
and the spores enumerated by the use of anaerobic plates after the method of 
Krumwiede. The suspensions were then diluted to give between 2,000,000 and 
4,000,000 spores per cc. 

The foods used were purchased from a local dealer and consisted of the 
following varieties:. fruits—apricots, apples, blackberries, cherries, goose- 
berries, peaches, pears, plums (green gage), black raspberries and strawberries ; 
vegetables—asparagus, red kidney beans, lima beans, beets, hominy, peas, dill 
pickles, sweet potato, pumpkin, sauerkraut and spinach; meats—salmon and 
shrimp, making 23 kinds of food in all. These were given a preliminary incu- 
bation at 37 C. for 30 days as a control for sterility, during which time only 
2 cans of sauerkraut showed any change, these becoming “springers” due, 
probably, to hydrogen. This incubation, of course, did not reveal the presence 
of thermophilic bacteria—those which cause “flat sours’ in certain foods. 
Since there were 18 cans of each kind of food, this indicates that the heating 
and processing of these 414 cans had probably been adequate so far as 
organisms which develop at 37 C. are concerned. 

Two cans of each food were then inoculated with each of the 4 spore 
suspensions, making 8 cans of each food in all. One set of 4 cans was held at 
room temperature for 10 months in a room at about 25 C.; the corresponding 
set was placed at 37 C. In all cases the cans were examined when they showed 
physical signs of spoilage by swelling, but otherwise they were incubated for 
the full 10 month period. The method of making the inoculation was as 
follows: The tops of the cans were scrubbed with alcohol and a small area 
was smeared with rosin flux. Through this area a hole was punched with 
a small nail and 1 cc. of the spore suspension introduced well into the center 
of the can with a glass syringe equipped with a long needle. The holes were 
then closed with solder. 

Tests for toxicity were made on each of the 184 cans inoculated by feeding 
approximately 0.5 cc. of the material to a guinea-pig with a pipet. When a 
strong toxin was present the animals died with typical symptoms in less than 
24 hours. In all cases the animals were kept under observations for 3 or 4 
weeks before a negative result was recorded. The results have been sum- 
marized in table 1. Of the 184 cans inoculated, 61 were toxic and 123 nontoxic. 
No more cans developed toxin at 37 C. than at room temperature, although 
the physical signs of spoilage were more evident at 37 C., since 49 cans at this 
temperature were apparently spoiled compared to 33 at room temperature. 
Much of this spoilage, which did not show toxin, judged by “swells” or 
“springers,” occurred in acid products such as gooseberries, cherries, straw- 
berries, sauerkraut, etc., in which an inflammable gas could be demonstrated, 
probably hydrogen. None of the fruits showed toxin production after the full 
incubation period. All the cans of beans, hominy, peas, sweet potato and shrimp 
were very toxic, and all cans of salmon except 2 were toxic. One can of 
pumpkin which was apparently normal contained a weak toxin which killed 
a guinea-pig in 17 days, the animal showing typical symptoms. In addition 3 
cans of pumpkins were spoiled, the contents of one being quite black, while 
but 2 were toxic. This would place pumpkin in the group of vegetables which 
might show irregular toxin production, a point which is not in agreement with 
the results obtained by Schoenholz, Esty and Meyer.’ 


6 L. THomMpson AND F. W. TANNER 

The hydrogen ion concentration of the foods was determined on one or more 
of the 10 cans which were kept as controls In certain cases both the poten- 
tiometer and the indicators of Clark and Lubs were used, the former being 
necessary when a highly colored liquid was to be tested as in beets, black- 
berries, raspberries and cherries. It was found that the two methods did not 
check exactly when tried on clear liquors from cans of spinach, asparagus, 
apples and apricots, but the variations in most cases did not amount to more 
than 0.2 of a point on the H-ion scale, and were no greater than the variations 
found in a series of cans of the same food when tested by the same method. 
The results of this determination of acidity on control cans are given in table 1. 
In addition, a number of determinations were made as follows: 8 cans of 


TAB TR 
RESULTS OF DETERMINATION OF ACIDITY ON CoNTROL CANS 

Room Temperature BiG. Total | Pu of 

Foods — ———<$—$___$___-___| T'ota]| Non-| Con- 
Average No. No. Average oO. No. | Toxic} Toxic} trol 

Incubation | Spoiled | Toxic| Incubation | Spoiled | Toxie 
IA PDICSIo eects cee 914 mos. 0 0 9144 mos 1 0) 0 8 3.6 
A DTICOUS tome sce nee 914 mos. 0) 0 94% mos 0 0 0 8 3.8 
Blackberries........ 914 mos. 0 0 914 mos 0 0 0 8 3.3 
Oherriesae. conto 914 mos 0 0 mos. 2: 0 0 8 3.4 
Gooseberries....... 914 mos 0 0 914 mos. 4 0 0) 8 3.0 
Peaches seo scthe se 10 mos 0) 0 10 mos 0 0 0 8 4.4 
POAT Rieti cirlels -brsine oe 10 mos. 0 0 10 mos 0 0! 0 8 4.2 
Plums (green gage)| 10 mos. 0 0 10 mos 1 0 0 8 3.6 
Raspberries (black)| 91% mos. 1 0 6 mos p: 0 0 8 4.3 
Strawberries........ 10 S=mos: 0 0 10 mos 3 0 0 8 apps 
ASPALaoUs: cee eter 9144 MOS. 1 i 914 mos 0! 0) 1 7 4.9 
Beans (red kidney) | 4% days 4 4 214 days 4 4 8 0 5.2 
IBeCans iM) em. ocr 2 days 4 4 2 days 4 4 8 0 5.6 
IBGCUS Me cctem hr s.0's 81% mos. 1 1 10 days- al 1 2 6 5.2 
10 mos 
Je loveabboa ye, oanmhcnas Ae 12 days 4 4 10 days 4 4 8 0 6.6 
POAS ater hirer nies arses 3 days 4 4 2 days 4 4 8 0 5.2 
Pickles (dill)........| 10 mos. 0: 0 10 mos z 0! 0 8 3.6 
Potato (sweet)..... 12 days 4 4 11 days 4 4 8 0 4.6 
Je(mnonosab RN esone nan. 914 mos. al 2 914 mos 2 1 3 5 5.0 
Sauerkraut... -e se 914 MOS. 1 0 7 mos 3 0 0 8 3.4 
SDIdelee cece see 9 mos. 0! 0 9 mos. 0) 1 1 tf 4.6 
SAIMLOUS, serctsce sce 4 days 4 3 2-39 days 4 3 6 2 6.6 
Shrintpercsceetee oe 5 days 4 4 4 days 4 4 8 0 6.8 
POLE rae meteytsiera sieve .« eeeenine 33 SLIM idsaeersde soe 49 30) 61 123 


spinach, in which the readings were 4.6 in six, and 4.8 in two; 9 cans of 
asparagus, in which the readings were 4.8 in six, 5.0 in two, and 5.6 in one. This 
suggests one possible explanation of why an occasional can of asparagus or 
spinach shows toxin production while the rest of the lot does not. The cans 
used appeared to be normal in every respect, and were evidently sterile, since 
they had been incubated for 30 days at 37 C. and held at room temperature 
for 10 months thereafter. The reason for this occasional variation in reaction 
will be discussed later. Four cans of gooseberries: which were hydrogen swells 
were selected from the controls and tested to determine what effect this had on 
reaction. Three cans gave a reading of 3.6 the same as that of a normal can, 
while one gave a reading of 4.2. In this case hydrogen swells did not seem 
to affect the reaction. 

The greatest interest in this study attaches to those foods which are 
irregularly toxic, such as asparagus, beets, pumpkin and spinach. With 
spinach, particularly, several investigators have had similar results—a few 


Toxin PRopUCTION BY CLOSTRIDIUM BOTULINUM 7 


cans of a lot made suitable medium while the remainder did not support 
growth. This failure to support growth has been correlated with the reaction 
by Schoenholz, Esty and Meyer,‘ who point out also that other conditions 
besides acidity are significant. They state also that the limiting acidity will 
vary with the kind of food. But with a given food. particularly with cans 
of the same lot, we look to the acidity as the chief factor, and in attempting 
to explain the variations found in cans of spinach a study of various kinds 
of natural medium was made. The object of using several kinds of canned 
foods as mediums was to obtain a comparative idea of their growth pro- 
moting abilities, and to be able to compare them with the best known artificial 
mediums. 

Accordingly, liquid culture tubes were made from peas, corn, white potato, 
salmon and spinach. When possible the liquor from the can was decanted and 
tubed along with fragments of the solid material, no attempt being made to 
use a filtered medium in any case. The potato medium was made from the 
fresh vegetable as follows: After peeling, the pulp was cut in small pieces, 
covered with water and boiled until it was soft. It was then strained through 
cheesecloth to remove most of the starch, and the liquid poured into tubes for 
use. At first the reaction was not adjusted in any of the mediums. After 
sterilization the mediums showed a fairly clear supernatant fluid and con- 
siderable sediment. The corn broth was opalescent, and the salmon broth had 
a layer of fat at the top. For comparison hormone broth, made according to 
Huntoon’s formula, and brain broth were used. These two have been found 
to give rapid and profuse growth with Clostridium botulinum. 


The results of numerous tests with these various kinds of mediums, 
using comparative methods, and heavy and light inoculations, with vege- 
tative cells, and with spores both young and old, make it apparent that 
brain broth, hormone broth, and potato broth are in the same class. All 
gave rapid and profuse growth, as evidenced by heavy clouding and gas 
production. It was a little surprising to find potato extract such a good 
medium. Growth in it seemed to be maintained at a high level for 
several days, longer in fact than in hormone broth, which began to show 
sedimenting and clearing by the third day. The potato broth in the same 
test was still turbid and giving off abundant gas on the fifth day. The 
-corn medium was found inferior to that made from peas, while the 
unneutralized spinach liquor did not support any visible growth. Potato 
broth would make a very useful medium for culture of Clostridium 
botulinum. 

When cultures of these various mediums were observed for ability 
to form spores, it was found that potato broth and corn broth, as well 
as hormone broth, were not well suited to sporulation. The latter was 
made with 0.25% dextrose, and the others naturally have an appreciable 
carbohydrate content. Any medium which produced large amounts of 
gas, expelling the paraffin caps from the tubes, did not give a satisfactory 
yield of spores. On the other hand, brain broth and the liquid from a 


8 L. THompson AND F. W. TANNER 


can of peas gave a very good yield with most cultures. The liquor from 
canned peas is especially useful, since it can be sterilized and the clear 
supernatant fluid used. This gives a good yield, and there is little débris 
to interfere. 

Since potato broth seemed to rank so highly as a growth producing 
substrate, several tests were made to discover the chemical nature of 
the medium. The only positive results were a strong test for starch, 
as was to be expected, and a small amount of amino nitrogen, about 
0.05%. This would make the total nitrogen at least 0.312%. The 
biuret reaction and tests for trytophane and reducing sugars were 
negative. 
| Attention was now directed to the spinach juice which, among the other 
natural mediums, was the only one which failed to show growth. A normal 
can was selected and the liquor removed aseptically. The H-ion concen- 
tration was about 4.5, using methyl red. It was adjusted to Px 7 and sterilized. 


Five days after inoculation there was no growth, and the control tubes showed 
that the medium had again been made acid by sterilization. In repeating the 


PAB eZ 
RESULT OF HEATING ON CANS OF SPINACH 


Vacuum | Physical Condition Pu Remarks 
Grade 1 0 Good; liquorcleatajncey. vaste ener 5.0 Seam not tight 
Grade 2 14 Soft, 1quo0r-cloudVeseen cree eee 6.8 
ce, 


Grade 3 | 23 Mushy, liquor dark and cloudy.... 


test, the medium was neutralized, heated in the autoclave, again neutralized 
and sterilized in ‘tubes. To a portion 0.5% glucose was added. In this case 
the reaction remained neutral after the sterilization, and abundant growth was 
secured after 2 days with a light inoculation. Growth was not long continued, 
however, and a test showed that the reaction in the inoculated tubes had again 
become acid, although the control tube, which was incubated with the others, 
showed little change, a point which will be referred to later. 
; From this experimentation it seems that spinach is also a good medium for 
Clostridium botulinum if it has a proper reaction, and that the irregular results 
from inoculating cans may in part be due to this factor. Attempts were now 
made to discover if possible why one can of spinach is too acid to support 
growth while another is a favorable medium. 

At this time the fresh spinach on the market was undergoing a soft slimy 
rot after standing for a day or two. Some spinach was purchased and carefully 
sorted into 3 grades as follows: (1) all sound leaves; (2) partly sound and 
partly decomposed leaves, about two-thirds good and one-third affected; 
(3) poor leaves, most of them affected with rot. 

A No. 2 can of each grade was packed and loosely sealed by means of the 
first roll on a hand closing machine. The cans were then heated at 5 pounds 
for 45 minutes and sealed while hot. They were then allowed to stand at room 
temperature for 2 or 3 days before they were examined. The results are given 
in table 2. As a check on the heating process the vacuum in the cans was 
determined, the gage readings being in inches. 


Tox1IN PRODUCTION BY CLOSTRIDIUM BOTULINUM 9 


This experiment showed that the reaction became increasingly alkaline 
in proportion to the amount of rotted spinach in the can. It showed 
further that a relatively small amount of poor spinach in a can might 
reduce the acidity to a point where Clostridium botulinum would grow 
and produce its toxin. 

On several different occasions the flora of rotted spinach obtained 
in the market was studied to determine the specific cause of the soft 
slimy rot. For the most part the predominant species were gram- 
negative nonspore-forming rods, producing a strong alkaline reaction in 
litmus milk and in sugar broths, showing that they were proteolytic 
rather than fermentative in action. The odor of decayed spinach indi- 
cates the presence of ammonia. 

We have then, perhaps, a rational explanation of why certain cans 
in a pack of spinach become toxic. A portion of poor spinach gets into 
a can occasionally, thus reducing the natural acidity. If spores of Clos- 
_tridium botulinum also happen to be introduced two conditions necessary 
for toxin production are fulfilled. In addition, it can be seen why phys- 
ical signs of spoilage do not always accompany toxin production. The 
work of Schoenholz, Esty, and Meyer,’ as well as our own, has shown 
that the growth of Clostridium botulinum in spinach causes an increase 
in acidity. When the initial reaction is near the limiting acidity, this 
increase due to the metabolism of the bacteria is sufficient to inhibit 
growth before any marked signs of spoilage have occurred, but not 
before there has been a considerable multiplication of cells. Since the 
medium is not well suited to spore formation, the cells formed will 
slowly autolyze and liberate toxin, and we should expect to find a 
stronger toxin after 5 months than after a week or 10 days, a fact 
which was noted by the authors named. Dozier '* has found that medi- 
ums with 1% of glucose are unfavorable for spore formation, and has 
correlated autolysis with toxin production. This would explain well the 
results of our observations on spinach. 

If we go back now to the results of the food inoculation we can see 
the relationship of toxin production to reaction.. In the case of aspara- 
gus, 8 cans were inoculated, and only one was toxic. Of the uninocu- 
lated controls, 9 were tested for reaction. Only one showed a reaction 
above Py 5.0, and that one was 5.6. It might not always happen that 
one can out of every 8 or 9 would show such a marked variation in 
reaction. This is illustrated by the results obtained with spinach. Of 


12 Jour. Infect. Dis., 1924, 35, p. 105. 


10 L. TuHompson AND F. W. TANNER 


the 8 cans inoculated, only one was weakly toxic after 8 months’ incuba- 
tion. None of the 8 control cans tested for reaction showed any marked 
change. Ifa larger series had been examined, no doubt one could have 
been found with a reaction less acid than Py 5. 


COMMENT 


The results of the inoculations of foods do not differ materially 
from those obtained by Schoenholz, Esty and Meyer.t The same gen- 
eral groups of foods were observed, each group consisting of about the 
same varieties. The foods used in this investigation and not by the 
foregoing authors are: apples, blackberries, gooseberries, dill pickles, 
red kidney beans, lima beans, hominy, and shrimp. The first 4 belong 
to the acid group of foods showing no toxin; the last 4 belong to the 
class of foods which are regularly and highly toxic after short incuba- 
tion, and which show marked physical signs of spoilage. The results 
of the inoculation of pumpkin place it in the intermediate group of foods 
which are irregularly toxic. This does not agree with the work cited, 
in which pumpkin is placed in the groups of foods which are regularly 
toxic. 

In the case of beets, the cans inoculated with culture Al were toxic, 
both at room temperature and at 37 C. No other cultures developed a 
toxin in beets. In this case it seems that beets were a more suitable 
medium for culture Al than for the other strains used, although it is 
possible that these cans may have been less acid in reaction than the 
others, and happened to be used with the same culture. 

The work on food inoculation in conjunction with that in which 
_various food extracts were used as medium serves to emphasize that 
Clostridium botulinum is not especially fastidious as to food require- 
ments, but grows well on a wide range of substances if a suitable reac- 
tion is present. In addition, certain foods, such as spinach, asparagus, 
beets, and string beans, which have a natural reaction near Py 5.0 in 
case a sound product is canned, have all the requirements for promoting 
good growth except perhaps acidity. If such foods were neutralized 
before inoculation, it should be possible to get consistent spoilage and 
toxin production. Since a large share of the outbreaks of botulism 
reported in this country have been caused by this group of foods, par- 
ticularly string beans and spinach, some procedure aimed at preserving 
the protective natural acidity of the food should be valuable. 


ToxIN PRODUCTION BY CLOSTRIDIUM BOTULINUM 11 


It is conceivable that the reaction in foods may be rendered less acid 
after canning by certain bacteria which escape the heating process, and 
thus make the material in the can suitable for the development of spores 
of Clostridium botulinum when they are present; this would amount to 
a bacterial metabiosis, and is less likely than that the alkaline substance 
is formed previous to canning, since the bacteria found in rotted vegeta- 
bles are often of the type producing an alkaline rather than an acid 
reaction on the common artificial mediums. 


. SUMMARY 


Twenty-three kinds of food were inoculated with detoxified spores 
of 4 strains of Clostridium botulinum. ‘Those found to be regularly 
toxic with all strains are: red kidney beans, lima beans, hominy, peas, 
sweet potato, salmon, and shrimp. Those irregularly toxic are: aspara- 
gus, beets, pumpkin, and spinach. The acid fruits and the very acid 
vegetable products, such as sauerkraut and dill pickles, did not become 
toxic. 

Certain extracts and liquors from canned vegetables were used as 
medium in comparison with brain broth and hormone broth. The 
extract from white potato was found equal to the best medium for pro- 
moting growth. The liquor from cans of peas, corn, and salmon gave 
lesser growth, while that from spinach gave growth only after being 
neutralized. | 

The reaction in cans of asparagus, string beans, beets, pumpkin, and 
spinach probably explains the irregularity of toxic production so fre- 
quently noted in these foods. 

The reaction in certain canned vegetables is greatly affected by the 
quality of the material put in the cans. Experiments with spinach show 
that a small amount of rotted material will reduce the acidity to a point 
where Clostridium botulinum can grow and develop a toxin. 


VITA 


EKDUCATION : 


B. A. Clark University 1916. M. A. University of 
Illinois 1928. 


PUBLICATIONS: 


A Group of Hydrogen Sulphide Producing Bacteria. 
Journal of Medical Research Vol. 42 (1921) p. 383 
Changes in Nutrient Agar Due to Clarifying with Ege. 
Journal of Laboratory and Clinical Medicine. Vol. 7 
(1922) p. 758 - 


TEACHING AND PROFESSIONAL EXPERIENCE: 


Instructor in Biology, Mercersburg Academy 1917-18. 
Instructor in Bacteriology, University of Pennsylvania 
1920-21. Assistant in Bacteriology, University of Illi- 
nois 1921-24. Assistant in Bacteriology, Mayo Founda- 
tion, University of Minnesota 1924-25. First assistant in 
Clinical Pathology, Mayo Clinic, 1924-25 


SOCIETIES: 


Society of American Bacteriologists, Society of Sigma Xi. 


ai 


0112 


LMM 
3 072884759 


